The repair and management of extensive burn wounds have long been a problem and challenge. Autografts are often used in the burn treatment;however, limited supply and the creation of secondary wound by harvesting autografts hold its wide application for extensive burns. Autologous tissue engineered skin substitutes (ATESS) are considered the promising alternative to autografts. They possess many of the properties of an autograft, such as wound protection, accelerated wound closure, high graft take, new skin regeneration, and no immunorejection. For extensive burn treatment using ATESS, there are two unsolved and critical issues: 1) the in vitro time to create skin substitutes should be as short as possible, allowing rapid wound closure;2) the size of ATESS should be as large as possible to cover enough wounded area. Current tissue- engineering approaches require prolonged culture time and cannot produce large skin substitutes. Therefore, it is desirable to develop new approaches and strategies toward rapid production of autologous skin substitutes with up-scaling potential. The primary objective of our research is to develop a novel practical protocol for rapid creation of ATESS using a nanofiber-enabled layer-by-layer cell assembly approach. Our specific aims are: 1) in vitro preparation and characterization of skin substitutes using layer-by- layer assembly of skin cells, 2) in vivo determination of the effect of tissue engineered skin substitutes on the healing of acute full-thickness wounds in a mouse model. These studies will help to develop a practical therapeutic protocol for acute or burn wound repair and regeneration. PUBLIC HEALTH RELEVANCE: This proposal is to establish an innovative approach to construct skin substitutes using layer-by-layer cell assembly with the assistance of nanofibers and evaluate the in vitro development of the assembled construct and its in vivo application for full-thickness wound repair. This approach allows us to rapidly (within one week or even overnight) create autologous skin substitutes of any size by directly assembling skin cells with biomimetic nanofibers during the collection of nanofibers. As a result, it will significantly shorten the in vitro time prior to in vivo implantation. The rapid creation of autologous skin substitutes will enable the quick closure of wound and, therefore, will be suitable for the treatment of extensive burn wounds. Additionally, this new approach can be easily adapted to tissue engineering of other types of layered tissues. This will accelerate the wide applications of tissue-engineered products in health care delivery.